Chemical compounds and processes for preparing the same



value in the treatment of various diseases. likely that Kendalls compound E and/or other closely related ll-hydroxyrsteroids will find increasing therapeutic CHEMICAL COMPOUNDS AND PROCESSES FOR PREPARING THE SAME Earl M. Chamberlin, Westfield, and John M. Chemerda,

Metuchen, N. J., assignors to Merck & Co., Inc., Rahway, N. J., a corporation of New Jersey Application December 2, 1955 Serial No. 550,776

1s Claims. ,(Cl. 260-3971) No Drawing.

This application is a continuation-in-part of our pending application, Serial No. 288,564, now abandoned, filed May 17, 1952, which in turn is a continuation-impart of Serial No. 240,051, now abandoned, filed August 2, 1951.

This invention is concerned with novel chemical compounds of the cyclopentanopolyhydrophenanthrene series, and processes for preparing the same; more particularly, it

pounds consisting of 3-hydroXy-7-keto-A unsaturated compounds and 3-hydroxy-7-keto-A usaturated compounds, and to processes for the preparation of such comuse in the future. Unfortunately,- the only method for the preparation of such compounds presently available utilizes desoxycholic or cholic acids as the starting material. Cholic and desoxycholic acids have hydroxy substituents in ring C at the 12-position, thus providing a means for introducing a functional substituent in the 11- position. However, cholic and desoxycholic acids which are obtained from animal bile are only available in limited amounts. Heretofore, no practical'method was available whereby a functional group could be introduced in ring C which will permit the use of more abundant steroids such as the sterols ergosterol, cholesterol, stigmasterol, or plant sapogenins such as diosgenin, tigogenin, and the like.

It is an object of the present invention to provide a process for introducing a functional group in ring B at the 7-position, which compounds can be converted to cortisone, by a process described in pending sole application of one of the present applicants, Serial No. 240,052, new U. S. Patent No. 2,734,897, filed August 2, 1951, which was filed simultaneously with our parent application Serial No. 240,051.

The compounds forming the subject matter of the present invention are believed to be represented by the formulae: 7 r

United States Patent relates to novel cyclopentanopolyhydrophenanthrene comi 0&

wherein R is either a sterol side chain (such as ergosterol), a bile acid side chain (such as cholanic acid), a degraded and bile acid side chain (such as bisnorcholanic acid), a'

pregnane side chain (COCH or a sapogenin side chain (such as tigogenin).

In accordance with our invention, these new compounds may be prepared by employing starting materials selected from the group consisting of hemi-epoxylated and hemi-hydroxylated steroids containing double bonds in the A", A or A901) positions; The hemi-epoxylated compounds having a double bond in the A or A901) position which may be employed as starting materials in our process, are believed to have in rings B and C the structure Patented. he: 411 958 "lhe hemihydroxylated compound having a double bond in the A' position which may be used as starting materials in our process are believedto have in rings B and C, the

structure r The hemi-epoxylated compounds which may be employed in our novel process may be indicated by the following complete formula: V

sq, I p

wherein R is as above, and R is hydrogen or an acyl group.

A compound of thetFormula IH or IIIA, wherein R is a bile acid sidechain (such as cholanic acid) and R' is an acyl group (namely, methyl-3-acyloxy-A choladienate epoxide) can be, prepared by reacting methyl- 3-acyloxyA"- -choladienate compounds with perbenzoic acid in a benzenemedium. The processes for preparing methyl-3-acyloxy-A -choladienate epoxide are disclosed and claimed in our co-pending application, Serial No. 215,026,1iled March 10, 1951. g

In: mdance with our' process, methyl-3 -acyloxy- A -chol'adienate epoxidej: c'anbe heated with acetic at temperatures of about150" C. to form prepared by reacting ergosteryl D acetate with perbenzoic acid in abenzene medium. exact structure of the epoxide is not known, and it is represented by the alternative Formula III or IIIA.) The processes for preparing ergosteryl D acetate epoxide (At present, the I (III or IHA) are disclosed and claimed in our co-pending application, Serial No, 11-5,026, filed March 10, 1951. Ergosteryl D acetate epoxide (IH or IIIA) is reacted I V with aqueous acidic reagents at a temperature ranging from 20-100 C. to form a mixture of 3-hydroxy-7-keto- A -ergostadiene (I) and 3'-hydroxy-7-keto-A ergostadiene (II). t

A variety of acidic reagents may be employed in this process, such as aqueous mineral acids (i. e., dilute hydrochloric or sulfuric acid), p-toluene sulfonic acid, oxalic acid, or aluminum nitrate hexahydrate. The reaction is preferably carried out in aqueous organic solvent medium, such as aqueous acetone, aqueous alcohol, or aqueous dioxane.

Instead of employing the acidic reagents mentioned above, we have found that the reaction can be carried out in water, but the reaction must then be conducted at higher temperatures, preferably at about 160 C.

The proportions of 3-hydroxy-7-keto-A -ergostadiene (I) and 3-hydroxy-7-keto-A -ergostadiene (II) present in the final product vary with the time, temperature and work-up of the reaction mixture. Usually at higher temperatures and/or longer reaction time, larger amounts of 3-hydroxy-7 keto-A --ergostadiene (I) are formed. Whereas, at lower temperatures and/or shorter reaction time, 3-hydroxy-7-keto-A -ergostadiene (II)? predominates in the mixture. Pure 3-hydroxy-7-keto- A -ergostadiene (I) may be recovered from the ture by dissolving the mixture in a 50% ether acetone solution and chromatographing the solution on an acid washed alumina. 3 hydroxy 7-keto-A -ergostadiene crystals separate from the eluate upon addition of suf ficient acetone.

The preferred procedure for converting the ergosteryl D acetate epoxide to the mixturesof ketones, 3-hydroxy- 7-keto-A -ergostadiene and 3 hydroxy 7 keto- A -ergostadiene, comprises refluxing the epoxide for about four hours in aqueous acetone or aqueousdioxane with a small amount of aqueous sulfuric acid, added as a 2 N aqueous solution. The acidity of the reaction mixture is usually in the rangegof a 0.2100001 normal acid solution. During. this rearrangement, at least apartial hydrolysis of the 3-acyloxy group of the starting material occurs, and additional treatment with alcoholic alkali achievescomplete' hydrolysis and leads to a mixture in which the compound 3-hydroxy-7-keto-A-"- ergostadiene ('I') is the predominant component; I. I The structure :ofthe hemi-hydroxylated compounds having a double bond in the A' or A901) position employed as starting :materials in? our process have not been definitely established; but are believed? to-be represented by the formulae B IVA- Compoun ds wherein R is hydrogen and R is a ste'rol side chain, such as in ergosterol, namely 3,7,8-1 1'jl1yn' -ergostadiene (IVA), are prepared by reacting ergosteryl 'DYacetate with osmium tetroxide and" the intermediate 'osm'at'e esters are hydrolyzed; such as by treatment with sodium sulfite, sodium bicarbonate,- or' aqueous mannitol.

The glycols thus formed, 3,7,8-trihydroxy-A ergostadiene or 5,9,ll trihydroxy-A' -ergostadiene are converted to 3-hydroxy-7-keto-A -ergostadiene and 3- hydroxy-7-keto-A3fi -ergostadiene, by treatment with aqueous acidic reagents. at a. temperature ranging from 20-l00 C. or by heating with water at C. in a sealed tube.

Our novel cyclopentanopolyhydrophenanthrene compounds consisting of 3-hydroxy-7-keto-M-unsaturated compounds and 3-hydroxy-7-keto-A -unsaturated compounds may also be prepared'from a hemi-hydroxylated steroid believed to be represented by the formula R r r I H0 i V n R'O OH wherein R and R are as above.

A compound of the above formula, wherein R is acetyl and R is a sterol side chain, such as in ergosterol (i. e., 3-acetoxy-7,ll-dihydroxy-n -ergostadiene) is reacted with sulfuric acid in a dioxane solvent. The reaction mixture is concentrated and water added. Whereupon a mixture of 3-hydroxy-7 keto-n -ergostadiene and 3-hydroxy-7-keto-A ergostadiene separates from solution and is recovered. t

A A -7 keto cyclopentanopolyhydrophenanthrene compound, a A -7-keto-cyclopentanopolyhydrophenanthrene compound or a mixture of these compounds, is reacted with an" acylating agent to produce the corresponding enol acylate, that is, the A -7-acyloxy-cyclopentanopolyhydrophenanthrene; the latter compound is treated with an organic peracid, whereby the double bond connecting the 9- and ll-carbon atoms is replaced by a 9,1l-epoxide linkage, thereby forming the corresponding A' -7-acyloxy-9,l1-epoxy cyclopentanopolyhydrophenanthrene. This latter compound is converted by reaction with an alcoholic solution of an alkali metal hydroxide to the corresponding A -7-keto-11-hydroxycyclopentanopolyhydrophenanthrene compound. This latter compound is reacted with an acylating agent'thereby forming A -7-keto-11-acyloxy-cyclopentanopolyhydrophenanthrene.

The A -3,11-dihydroxy 7 keto cyclopenta'nopolyhydrophenanthrene compounds are readily converted to steroid hormones having an oxygen atom attachedto the ll-carbon atom as substantially described in U. S. Serial No. 240,052 as follows: The A -3,11-dihydroxy-7-ketohydrophenanthrene compound, which is heated withwa diethylene glycol solution of hydrazine and potassium hydroxide to produce the corresponding 3,l1-dihydroxycyclopentanoperhydrophenanthrene compound; this 3,11- dihydroxy-cyclopentanoperhydrophenanthrene compound is then treated according to known methods (the ex'act method of treatment depending upon the side chain attached to the 17-carbon atom) to convert the organic substituent attached to the 20-carbon atom to a keto radical thus forming the corresponding 3,11-dihydroxy-20- keto-pregnane. (For example, where the side chain attached to the 17-carbon atom is that present in the bile acid, bisnorallocholanic acid, the 3,11 dihydroxyrbisnorallocholanic acid is esterified, the methyl 3,1l-dihydroxy-bisnorallocholanate is reacted with phenyl magnesium iodide to form 3,1l-dihydroxy-20-allopregnanyldiphenylcarbinol; the latter compound is reacted with acetic anhydride to produce 3,1l-diacetoxyallo-etiocholanyl methyl dipenyl ethylene, and this vcompound. is treated with ozone to form 3,1l-diacetoxy-20 keto-allopregnane.) This 3,1l-diacetoxy -2O keto-allopregnane (obtained as described above starting with'3,l1-dihydroxybisnorallocholanic acid, or if preferred by degrading the side chain of some other 3,1l-dihydroxy-cyclopentanoperhydrophenanthrene compound) is thenreactedxwith an acetic anhydride in the presence of p-toluene sulfonic acid catalyst thereby forming the corresponding A 3,1l,20-triacetoxy-pregnene; this compound is reacted with perbenzoic acid followed by a hydrolyz ing agent to form 3,11,17-trihydroxy-20-keto-allopregnane; the latter compound is then reacted with bromine followed by sodium acetate thereby forming the corresponding 3,11,17- tn'hydroxy-ZO-keto-Zl-acetoxy-allopregnane; the 1 3,11,17- trihydroxy-ZO-keto-Zl-acetoxy allopregnane is then reacted with chrornic acid thereby forming 3,11,20-triketo-l7- hydroxy-Zl-acetoxy-allopregnane which is reacted'with two molecular equivalents of bromine followedby rearrangement to produce the corresponding 2,4 dibrom o- 3 ,1 1,20-triketo-17-hydroxy-2l-acetoxy-allopregnane; this compound is reacted with a dehydrohalogenating agent and a reducing agent to form cortisone acetate, i.,e. A -3 1 l ,20-triketol7-hydroxy-2 l-acetoxy-pregnene.

The following examples are presented to illustrate the specific embodiments of our invention:

EXAMPLE 1 cyclopentanopolyhydrophenanthrene compound is reacted with zinc and acetic acid thereby forming the corresponding 3,11-dihydroxy 7 keto cyclopentanopolystirring at 5 C. to a solution containing 4.28 g; (0.01

OOCH:

OOCHI i o o OCH: o

' Twenty-six cc. of a benzene solution of perbenzoic acid (0.011'mo1) was added over a period of one hour with u 7 V a Alleltflltiition was'complete, the solution; was stirred -fdr one -halb hour: at Ca and then for. three hours at mitm emmre. Onemolar equivalentof perbenzoic acidawalconzumed. V Tliet'benzenesolution was washedwith 3 portionsot 30 ca ol-a cold 5% potassium hydroxide solution to remove 1h mcidual: perbenzoid acid" and other. acidic material {mm by ice water until neutral. The neutral benzene solution wa'e: dried over anhydrous. sodium sulfate and conecntrBte'diu-vacuo. A yield of 4.3 g; of crude methyl G wetOXy-N' -chOladienate epoxide melting at l42.6 obtained. Thisprocedure is: fully descr ibed and dfimeddn our co-pending'application, Serial No. 215,026, fled Ma'ch 10,-1-951. 7

of methyl-3'-acetoxy-A"- -choladienate epoxideprepared as above were suspended in 20 cc. of anhydnide', and the suspension was heated in a tube at a-temperature of 150 C. fora periodof about fourlrours. The reaction solutionwas cooled to whereupon large rectangular crystalliileiplated separated from the solution. 1 This crystalline materiel was recovered'by filtration, washed with acetic iuhydrideand-d'ried to give about 1.1 g. of crude methyl- 3-aeetoxy-7--keto=A -cholenate; having a melting point of 168-170 C. This material was purified by chromatographyfollowed by recrystallization from methanol to give substantially pure methyl-3-acetoxy-7-keto-A -cholenate, havinga melting point of 185 C.

EXAMPLE 2 Alternative procedure for the preparation of methyl-3- acetaxy- 7: keto -A" choladienate from methyl-3- dcetoky- A' -chaladienate epoxide once from acetone to give substantially .pure methyl-3- acetoxy-Fket'o-A" -cholenate; having a meltingpoint of EXAMPLE 3 'Ergoste'ryL1D; acetate. epoxide, the starting. material employed int-this-reaction, was prepared by dissolving. 1' gram of ergosteryl D acetate in 30 cc. of benzene and adding 10 cc. of perbenzoic acid solution equivalent to 1.21 millimols of oxygen. A-fter five minutes, the perbenzoic acid was consumed and the reaction mixture was transferred to a separatory funnel with cc. of benzene. The solution was washed four times with 25 cc. of 1% potassium hydroxide solution andthen with water until the washings were neutral. The benzene solution was dried over anhydrous'sodium sulfate, filtered and the solvent removed in vacuo under nitrogen. The crude ergosteryl D acetate epoxide on recrystallization from acetone melted at 208-210" C. The procedure for preparing ergosterylvD acetate epoxide is fully describedand claimedinour coending application, Serial No. 215,026, filed March 1:0, 1 95 l. Five hundred milligrams of ergosteryl D acetate epoxide prepared as described above was suspended in 50 cc. of acetone. The suspension was stirred and 1 cc. of a 2:N sulfuric acidsolution added, whereupon a clear solution resulted. The solution was allowed to stand for eighteen'hours'atroomv temperature, thenl cc. of). 2.5

sodium hydroxide was added and the mixture concentrated in vacuo-.; The residue had a melting point of 1-65-468" C.,- and ultraviolet absorption examination of the residue indicated that 3 hydroxy-7-keto-A -ergostadiene was present to the extent of -80%; A max. 2440-2460 A-., 15% 162 (isooctane solvent). The remaining 40-20% was 3-hydroxy-7-keto-A -ergostadiene.

V EXAMPLE 4 Preparation of 3-hydr0xy-7-ket0-A -erg0stadiene having a meltingpoint of l42-1 47 C.

Purification was accomplished by dissolving the crude 3-hydroxy-7-keto-A -ergostadicne in ethyl ether and subjecting this solution to chromatography on acid washed alumina using an acetone-ether mixture for elution. The

Preparation of'a mixture of 3-hydroxy 7-keta-A -ergostadiene and 3-hydroxy-7-keto-A -erg0stadiene from ergosteryl D acetate epoxide HtO-acetone 9 product thus obtained was further purified by a second chromatography using a mixture of methanol-ether for elution. v

The purified 3 hydroxy 7 keto A -ergostadiene melted at 1535-1545 C., had a specific rotation EXAMPLE 5 Preparation of a mixture of 3-hydroxy-7-ket0A -ergostadiene and 3 hydroxy 7 keto A -ergostadiene from ergosteryl D acetate epoxide To a mixture of three grams of ergosteryl D acetate epoxide, 120 cc. of dioxane and 30 cc. of water was added 6 cc. of 2 N sulfuric acid. The resulting cloudy mixture was heated to 80-90 C. for five minutes, whereupon the mixture became clear. The clear mixture was al lowed to stand at room temperature for twelve hours and crystals in the form of needles separated from solution. The crystals which were recovered weighed 940 milligrams, had a melting point of 172-180" C., and con- 'sisted principally of 3-hydroxy-7-keto-A p' ergostadiene together with a small amount of 3-hydroxy-7-keto- A -ergostadiene as indicated by the ultraviolet absorption A max. 2440 A.,E1, 4.l6 (isooctane solvent) A max. 375 m E1 1, 356 (in chloroform) EXAMPLE 6 Preparation of 3-hya'roxy-7-keto-A -ergostadiene and 3-acetoxy-7-keto-n -ergostadiene from ergosteryl D acetate epoxide Five hundred milligrams of ergosteryl D acetate epoxide was dissolved in 20 cc. of hot dioxane and the mixture diluted with 5 cc. of water. To the cloudy reaction mixture was added 1 cc. of 2N sulfuric acid solution,'whe reupon the solution cleared. The reaction mixture was heated for twenty-four hours and then concentrated upon the addition of water. The solid product, a mixture of 3 hydroxy 7 keto A ergostadiene' and 3 hydroxy-7-keto-A -ergostadiene crystallized from solution. The crystals which were recovered melted at 148- 157 C. and had an ultraviolet absorption A max. 2540 A, E% 150-180 (alcohol solvent).

Pure 3-hydroxy-7-keto-A -ergostadiene was obtained by dissolving the mixture in a 50% ether-acetone solution and chromatographing the solution on 15 grams of acidwashed alumina. From the 50% ether-acetone eluate,

'3 hydroxy-7-keto-A -ergostadiene was obtained by crystallization from acetone. The 3-hydroxy-7-keto-A 'ergostadiene thus obtained possessed the physical characteristics:

Melting point: 179180 C. Ultraviolet absorption:

A max. 246 m EZ 240 (isoootane solvent) An alysis.Calculated for 0 mm,: 0, 81.52; H,

10.74. Found: C,81.53; H, 10.67. 3-hydroxy-7-keto-A -ergostadiene .was reacted, with boiling acetic anhydride to yield 3-acetoxy-7f-k eto-A ergostadiene which was further purified by crystallization from methanol-acetone as needles:

Melting point: 21-3213.5 C.

Specific rotation: a 58.6 (1.19% in chloroform).

' Calculated for C H O C, 79.24; H, 10.20. Found:

C, 79.81; H, 10.10. t

EXAMPLE 7 Preparation of 3-hydroxy-7-ket0-A -ergostadiene' One hundred grams of ergosterol D epoxide was suspended in 5 liters of absolute ethanol. To the suspension was added 500 cc. of 2 N sulfuric acid. The reaction mixture was then refluxed in a nitrogen atmosphere for three hours and 6 liters of ice water was added. The reaction mixture was filtered, the residue washed free of acid with water, and air dried. The crude product was dissolved in ethyl ether and chromatographed on acid washed alumina. From the methanol ethyl ether eluate was obtained substantially pure 3-hydroxy-7-keto-A ergostadiene.

EXAMPLE 8 Preparation of 3-hydroxy-7-keto-n -ergostadiene and 3-acetoxy-7-keto-A -ergostadiene from ergosteryl D acetate epoxide One and two-tenths grams of ergosteryl D acetate epoxide in 50 cc. of dioxane and 5 cc. of water was heated at C. in a sealed tube for sixteen hours. Concentration of the reaction mixture and dilution with water yielded a mixture of 3-hydroxy-7-keto-A -ergostadiene and its 3-acetoxy derivative, 3-acetoxy-7-keto- A -ergostadiene, which was separated conveniently by chromatography over alumina. J

EXAMPLE 9 Preparation of a mixture 0 3-hydr0xy-7-ket0-A -ergostadiene and S-hydroxy 7 keto-A -erg0stadiene from ergosteryl D acetate epoxide EXAMPLE 10 Preparation of a mixture of 3-hydr0xy-7-ket0A -ergostadiene and 3-hydr0xy 7 ket0-A ergostadiene from ergosteryl D acetate epoxide sodium sulfate and concentrating to dryness, a crystalline residue consisting of a mixture of 3-hydroxy-7-keto-A ergostadiene and 3-hydroxy-7-keto-A -ergostadiene was obtained which was recrystallized from alcohol.

This mixture possessed the following physical characteristics:

Melting point: 163-165 C.

3-hyd roxy-7-keto-A b -ergostadiene from ,i-ace-toxy- 7,1 1-dihydroxy-A -ergostadiene no r - The starting material. in the reaction, 3-acetoxy-7,l1- dihydroxy-A -ergostadiene, was'prepared by dissolving 309 grams of ergosteryl D acetate in 1876 cc. of benzene and oxidizing at -15" C. with 107.6 grams of perpenzoic acid in 1775 cc. of benzene. The reaction mixture wasallowed to stand overnight. It was washed twice with 630 cc. ofa 5% potassium hydroxide solution, three times with 1000 cc. of water and dried over anhydrous magnesium sulfate.

Seventy. cubic centimeters of the above solution was diluted with an additional 50 cc. of benzene and 100 grams of'acid washed alumina was added. After standing. four'days, the alumina was filtered off and washed asifollowsr 1000 benzene at'room temperature 6x300 cc.: methanol at room temperature 500cc. boilingmethanol.

500 cc. boiling chloroform' The product from the cold methanol washes was taken up hot methanol/chloroform and insoluble material was removed by filtration: On concentrating the filtrate to a small volume and seeding, 820 mg. of. B-acetoxy- 7,11dihydroxy-A -ergostadiene (M. P. 238.5240.5 C.) was obtained. 7 i

A suspension of 300 milligrams of 3-acetoxy-7,11- dihydroxy-8,22-ergostadiene in cc. of dioxane and 2 cc.: of: 2 N sulfuric acid was refluxed four hours. The mixture: was; concentrated to. a small volume, andi water added, whereupon a mixture of:3-hydroxy-7-keto-M' ergostadiene and 3-hydroxyw7-ketoA -ergostadiene separated; from. solution. The precipitate was recovered by filtration After. chromatography over alumina, pure 3-hydroxyr7-ketorA -ergostadiene. was isolated upon re-. crystallization from methanol. The pure 3.-hydroxy-7- heto-A'fi ergostadiene. thus. recovered. .had a. melting of l80-.-18.1. C. andv exhibited. an ultraviolet. absorption r max. 246 m E2... 2l3 -(isooetane solvent) 12 EXAMPLE 12 Preparation of '3,9,1Iflrihydroxy-A -ergostadiene or 3,7,8-trihydroxy-A -erg0stadiene from ergosteryl D acetate To 508 milligrams of osmium tetroxide in 25 cc. of ether was added 876 milligrams of ergosteryl D acetate together with four drops of pyridine. After standing for four days the. reaction mixture was concentrated in vacuo. To the residue was added 15 cc. of 5% sodium hydroxide .in methanol and 0.5 g. of mannitol and the mixture refluxed for fifteen minutes.

The reaction mixture was diluted with 35 cc. of'water, whereuponthecrude product separated from solution. Two recrystallizations from acetone yielded pure 3,9,11- trihydroxy A7122 ergostadiene (or 3,7,8 trihydroxy- A -ergostadiene) having a. melting point of 194- 195 C. Analysis indicatedthat the product was solvated with one mole of acetone.

Calculated for C28H4603,CH3COCH3: F. H, 10.72.. Found: @7616; H, 10.40.

This compound was reacted with acetic anhydride and pyridine at room temperature to yield the corresponding diacetoxy derivative, 3,1 1-diacetoxy-9-hydroxy-A' -ergostadiene or 3,7-diacetoxy-8-hydroxy-A -ergostadiene (having a melting point of l62.5163 C.) from which the free hydroxy compound could be regenerated by hydrolysis.

Calculated for C H O C, 74.61; H, 9.79. Found: C, 74165; H, 10.02.

EXAMPLE 13 Preparation of 3,9,11-trihydroxy-A erg0stadiene' or 3 57,8-trihydroxy-A -ergostadiene from ergosteryl D acetate A solution of 8.8 grams of ergosteryl D acetate in 150 cc. of ether and 4 cc; of pyridine was treated with 5.1 gramsxof. osmium tetroxide. After three days at room temperature, the dark reaction mixture containing solid osmate ester, was stirred with cc. of 2.5 N sodium hydroxide containing 5.0 grams of mannitol. for 16 hours. During this period, the ether evaporated and a practically colorless, crystalline product wasobtained, which consisted of 3-acetoxy-9,llrdihydroxy-A -ergostadiene 0r 3-acetoxy-7,8-dihydroXy-A -crgostadiene and the-corresponding trihydroxy compound, 3,9,11-trihydroxy-Al -ergostadiene (or 3,7,8-trihydroxy-A ergostadiene) Chromatography of the crude product over" alumina yielded the pure crystalline 3-acetoxy-compound. aswell as some of the expectedtrihydroxy compound. The residue fi'dm'zthe ether -20%' acetone eluate crystallized from acetone to give needles of 3-acetoxy-9,11-dihydroxy A7'??- ergostadiene or' 3-acetoxy-78-dihydroxy-A -ergsta diene, having a melting point of 171.5-175 C. h

Calculated for G l-1 0 C, 76.22; -H. 10.24.; Found: C, 76.26; H, 10.05.

' This product reacted with acetic anhydride-pyridine to give a diacetoxy compound identical with the product obtained in Example 12, 3,11-diacetoxy-9-hydroxy-A" ergostadiene or 3,7-diacetoxy-8-hydroxy-A -ergostadiene, and had a melting point of 162.1-163 C.

EXAMPLE 14 Preparation of 3-hydr0xy-7-ket0-A -ergostadiene and 3- hydroxy-7-keto-A -ergostadiene from 3,7,8-trihydroxy-A -ergostadiene (or 3,9,11-trihydroxy-A ergostadiene) UiI One hundred milligrams of 3,9,11-trihydroxy-A -ergostadiene (or 3,7,8,-trihydroxy-A -ergostadiene) was heated with 4 cc. of dioxane, 1 cc. of water and 0.2 cc. of 2 N sulfuric acid on the steam bath for 2 hours. The mixture of 3-hydroxy-7-keto-A -ergostadiene and 3-hydroxy-7-keto-A -ergostadiene melted at 151-157 C., and exhibited an ultraviolet adsorption A max. 246 m Efi 188 (isooctane solvent) and wherein R is selected from the group consisting of an ergosterol side chain, a cholanic acid side chain and an esterified cholanic acid side chain and R is selected from the group consisting of acyl and hydrogen.

2. 3,7,8-trihydroxy-A -ergostadiene.

3. 3,9,11-trihydroxy-A -ergostadiene.

4. 3,7-diacetoxy-8-hydroxy-A -ergostadiene.

5. 3,1 1-diacetoxy-9-hydroxy-A -ergostadiene.

6. A compound selected from the group consisting of 3-acetoxy-7,8-dihydroxy-A -ergostadiene and 3-acetoxy-9,1 l-dihydroxy-A" -ergostadiene.

7. The process which comprises reacting ergosteryl D acetate with osmium tetroxide to form the osmate ester of ergosteryl D acetate, hydrolyzing the latter compound to form 3,9,11-trihydroxy-A' -ergostadiene reacting the latter compound with aqueous acidic acid to form a mixture of 3-hydroxy-7-keto-A -ergostadiene and 3-hydroxy- 7-keto-A -ergostadiene.

8. The process which comprises reacting ergosteryl D acetate with osmium tetroxide to form the osmate ester of ergosteryl D acetate, hydrolyzing the latter compound to form 3,7,8-trihydroxy-A ergostadiene reacting the latter compound with aqueous acidic acid to form a mixture of 3-hydroxy-7-keto-A -ergostadiene and 3- hydroxy-7-keto-A -ergostadiene.

9. The process which comprises reacting ergosteryl D acetate with osmium tetroxide to form the osmate ester of ergosteryl D acetate, and hydrolyzing the latter compound to form 3,9,11-trihydroxy-A' -ergostadiene.

10. The process which comprises reacting ergosteryl D acetate with osmium tetroxide to form the osmate ester of ergosteryl D acetate, and hydrolyzing the latter compound to form 3,7,8-trihydroxy-A -ergostadiene.

11. The process which comprises reacting 3,9,11-trihydroxy-A"' -ergostadiene with aqueous acidic acid to form a mixture of 3-hydroxy-7-keto-A -ergostadiene and 3-hydroxy-7-keto-A ergostadiene.

12. The process which comprises reacting 3,7,8atrihydroxy-A -ergostadiene with aqueous acidic acid to form a mixture of 3-hydroxy-7-keto-A -ergostadiene and 3-hydroxy-7-keto-A -ergostadiene.

13. The process which comprises reacting a compound of the formula to form the osmate ester, hydrolyzing the osmate ester to form a mixture of the following compounds R HO 1 HO n'o I V 2,840,532 5 ,16 wherein R isas above and Rf is selected from the group a consisting'offa'cyl andhydrogen; and'separating theabove mixture.

7 References Cited-inthe' file-ofthis' patent' UNITED STATES PATENTS Re: 22,941 Reichstein Nov. 25, 1947 

1. TRIHYDROXY COMPOUND SELECTED FROM THE GROUP CONSISTING OF 